Enhancing Wound Healing and Anti-Inflammatory Effects by Combination of CIGB-258 and Apolipoprotein A-I against Carboxymethyllysine Toxicity in Zebrafish: Insights into Structural Stabilization and Antioxidant Properties.

CIGB-258 is known to exert anti-inflammatory activity via structural stabilization of apolipoprotein A-I (apoA-I) and functional enhancement of high-density lipoproteins (HDL) against acute toxicity of carboxymethyllysine (CML). The co-presence of CIGB-258 in reconstituted HDL (rHDL) formed larger rHDL particles and enhanced anti-inflammatory activity in a dose-dependent manner of apoA-I:CIGB-258, 1:0, 1:0.1, 1:0.5, and 1:1 of molar ratio, in the synthesis of the rHDL. However, no study has evaluated the enhancement of HDL functionality by the co-presence of lipid-free apoA-I and CIGB-258. The present study was therefore designed to compare the structural stabilization and functional improvement of HDL in the presence of lipid-free apoA-I and CIGB-258 in molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1 within both HDL2 and HDL3. As the concentration of CIGB-258 increased, it effectively inhibited the cupric-ion-induced oxidation of HDL, thereby safeguarding apoA-I from proteolytic degradation. Additionally, the wound-healing activity of zebrafish was significantly (p < 0.01) enhanced by the co-addition of apoA-I:CIGB-258 (1:1) up to 1.6-fold higher than apoA-I alone (1:0) under the presence of CML. ApoA-I:CIGB-258 (1:1) treatment exhibited the lowest apoptosis and production of reactive oxygen species against CML-induced damage in the wound site. Also, an increase in wounded tissue granulation and epidermis thickness was observed with increasing concentration of CIGB-258 during 48 h post-treatment via the healing process. Intraperitoneal injection of apoA-I:CIGB-258 mixture remarkably ameliorated the acute paralysis and restored zebrafish swimming ability impaired by the acute toxicity of CML. The increase of CIGB-258 content, especially co-injection of apoA-I:CIGB-258 (1:1), leads to a significant 2.3-fold (p < 0.001) and 4.1-fold (p < 0.001) higher zebrafish survivability and recovery of swimming ability, respectively, than those of CML-control. In the apoA-I:CIGB-258 (1:1) group, neutrophil infiltration and interleukin (IL)-6 production was lowest in the hepatic tissue with the least cellular damage and apoptosis. Additionally, the group treated with apoA-I:CIGB-258 (1:1) demonstrated the lowest plasma levels of total cholesterol (TC) and triglycerides (TG), along with minimal damage to the kidney, ovary, and testicular cells. Conclusively, co-treatment of CIGB-258 with apoA-I effectively mitigated acute inflammation in zebrafish, safeguarded vital organs, structurally stabilized apoA-I, and enhanced HDL functionality.

Comparative Assessment of Beeswax Alcohol and Coenzyme Q10 (CoQ10) to Prevent Liver Aging, Organ Damage, and Oxidative Stress in Hyperlipidemic Zebrafish Exposed to D-Galactose: A 12-Week Dietary Intervention.

The current study was designed to compare in vivo efficacy between beeswax alcohol (BWA) and coenzyme Q10 (CoQ10) to treat fatty liver changes, oxidative stress, and damages in major organs of zebrafish by 12 weeks with high-cholesterol (HC) and galactose (Gal) supplementation. At week 12, the HC control and HC+Gal control groups showed 96% and 92% survivability, respectively, while co-supplementation of the 0.5% BWA and 1.0% BWA groups exhibited 96% and 100% survivability. However, co-supplementation of the 0.5% CoQ10 and 1.0% CoQ10 groups revealed the lowest survivability, around 92% and 89%, respectively. The 0.5% BWA and 1.0% BWA groups showed 21% (p < 0.001) and 41% (p < 0.001), respectively, lower total cholesterol (TC) than the HC+Gal control, while the 1.0% CoQ10 group showed only 15% lower TC than the control. Interestingly, the 0.5% BWA and 1.0% BWA groups showed 22% (p < 0.001) and 38% (p < 0.001), respectively, lower triglyceride (TG) than the HC+Gal control. However, both the 0.5% CoQ10 and 1.0% CoQ10 groups showed similar TG levels as the control, suggesting that CoQ10 supplementation had no effect on lowering serum TG. The 1.0% BWA group showed the highest plasma HDL-C and HDL-C/TC (%) up to 3.2-fold and 5.5-fold, respectively, higher than those of the HC+Gal control, while the 1.0% CoQ10 group showed 2.4-fold and 2.8-fold higher plasma HDL-C and HDL-C/TC (%), respectively, than the control. The plasma aspartate transaminase (AST) and alanine transaminase (ALT) levels were lowest in the 1.0% BWA group, 51% and 72%, respectively, lower than HC+Gal control, suggesting the lowest extent of hepatic damage. In hepatic tissue, neutrophil infiltration and interleukin (IL)-6 production were the lowest in the 1.0% BWA group, around 67% and 85%, respectively, lower than the HC+Gal control. Fatty liver change, cellular apoptosis, and cell senescence in hepatic tissue were remarkably lowered in the 1.0% BWA group, while the CoQ10 group showed much less effect than the BWA group. In kidney, ovary, and testis tissue, the 1.0% BWA group showed the lowest production of reactive oxygen species, the extent of cellular senescence, and cellular apoptosis with the healthiest cell morphology. In conclusion, supplementation of BWA remarkably protected the liver, kidney, ovary, and testis from oxidative damage by cholesterol and galactose consumption, with the least serum AST and ALT levels, inflammatory parameters, and senescence markers.

Comparison of the In Vivo Efficacy of Cuban (Raydel®) and Chinese (BOC Science) Policosanol in Alleviating Dyslipidemia and Inflammation via Safeguarding Major Organs and Reproductive Health in Hyperlipidemic Zebrafish: A Twelve-Week Consumption Study.

Policosanol is a blend of long-chain aliphatic alcohols (LCAAs) and is well-known for several health-beneficial activities; however, the functionality of policosanol varied substantially based on the composition of LCAAs. In this study, two distinct policosanols, Raydel® (extracted from Cuban sugarcane wax) and BOC Sciences (extracted from Chinese sugarcane wax), were dietarily supplemented (0.1% w/w) for 12 weeks in hyperlipidemic zebrafish to examine their influence on the blood lipid profile and functionality of the liver, kidney, and reproductive organs. The results demonstrated a noteworthy impact of both policosanols on preventing high-cholesterol diet (HCD, 4% w/w)-induced dyslipidemia by decreasing total cholesterol (TC) and triglyceride (TG) levels in the plasma. However, compared to BOC Sciences, the Raydel® policosanol exhibited a significantly (p < 0.05) higher efficacy in reducing HCD-induced TC and TG levels. A substantial effect was observed exclusively with the Raydel® policosanol in mitigating HCD-impaired low-density-lipoprotein cholesterol (LDL-C) and high-density-lipoprotein cholesterol (HDL-C) levels. Hepatic histology and immunohistochemistry (IHC) analysis revealed the higher efficacy of Raydel® policosanol over BOC Sciences policosanol to prevent HCD-provoked fatty liver changes, cellular senescence, oxidative stress, and interleukin (IL)-6 production. Consistently, a significantly higher effect of Raydel® over BOC Sciences policosanol was observed on the protection of kidney, testis, and ovary morphology hampered by HCD consumption. In addition, Raydel® policosanol exhibited a notably stronger effect (~2-fold, p < 0.05) on the egg-laying ability of the zebrafish compared to policosanol from BOC Sciences. Furthermore, Raydel® policosanol plays a crucial role in improving embryo viability and mitigating developmental defects caused by the intake of an HCD. Conclusively, Raydel® policosanol displayed a substantially higher efficacy over BOC Sciences policosanol to revert HCD-induced dyslipidemia, the functionality of vital organs, and the reproductive health of zebrafish.

Efficacy Assessment of Five Policosanol Brands and Damage to Vital Organs in Hyperlipidemic Zebrafish by Six-Week Supplementation: Highlighting the Toxicity of Red Yeast Rice and Safety of Cuban Policosanol (Raydel®).

Policosanol is a mixture of long-chain aliphatic alcohols (LCAAs) derived from various plant and insect origins that are marketed by various companies with distinct formulations and brand names. Policosanols offer several beneficial effects to treat dyslipidemia and hypertension; however, a comprehensive functionality comparison of various policosanol brands has yet to be thoroughly explored. In the present study five distinct policosanol brands from different origins and countries, Raydel-policosanol, Australia (PCO1), Solgar-policosanol, USA (PCO2), NutrioneLife-monacosanol, South Korea (PCO3), Mothernest-policosanol, Australia (PCO4), and Peter & John-policosanol, New Zealand (PCO5) were compared via dietary supplementation (1% in diet, final wt/wt) to zebrafish for six weeks to investigate their impact on survivability, blood lipid profile, and functionality of vital organs under the influence of a high-cholesterol diet (HCD, final 4%, wt/wt). The results revealed that policosanol brands (PCO1-PCO5) had a substantial preventive effect against HCD-induced zebrafish body weight elevation and hyperlipidemia by alleviating total cholesterol (TC) and triglycerides (TG) in blood. Other than PCO3, all the brands significantly reduced the HCD's elevated low-density lipoprotein cholesterol (LDL-C). On the contrary, only PCO1 displayed a significant elevation in high-density lipoprotein cholesterol (HDL-C) level against the consumption of HCD. The divergent effect of PCO1-PCO5 against HCD-induced hepatic damage biomarkers, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), was observed. PCO1, PCO2, and PCO4 efficiently curtailed the AST and ALT levels; however, PCO3 and PCO5 potentially aggravated the HCD's elevated plasma AST and ALT levels. Consistently, the hepatic histology outcome revealed the least effectiveness of PCO3 and PCO5 against HCD-induced liver damage. On the contrary, PCO1 exhibited a substantial hepatoprotective role by curtailing HCD-induced fatty liver changes, cellular senescent, reactive oxygen species (ROS), and interleukin-6 (IL-6) production. Likewise, the histological outcome from the kidney, testis, and ovary revealed the significant curative effect of PCO1 against the HCD-induced adverse effects. PCO2-PCO5 showed diverse and unequal results, with the least effective being PCO3, followed by PCO5 towards HCD-induced kidney, testis, and ovary damage. The multivariate interpretation based on principal component analysis (PCA) and hierarchical cluster analysis (HCA) validated the superiority of PCO1 over other policosanol brands against the clinical manifestation associated with HCD. Conclusively, different brands displayed distinct impacts against HCD-induced adverse effects, signifying the importance of policosanol formulation and the presence of aliphatic alcohols on the functionality of policosanol products.

Oral Supplementation of Ozonated Sunflower Oil Augments Plasma Antioxidant and Anti-Inflammatory Abilities with Enhancement of High-Density Lipoproteins Functionality in Rats.

Research on ozonated sunflower oil (OSO) is mostly restricted to its topical application, whereas the functional and toxicological assessment of oral OSO consumption is yet to be solved. Herein, OSO was orally supplemented in rats to assess the impact on plasma antioxidant status, low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Also, the functionality of HDL from the OSO-supplemented rats (OSO-HDL) was tested against carboxymethyllysine (CML)- induced hyperinflammation in embryo and adult zebrafish. The results revealed that four weeks of OSO supplementation (3 g/kg BW/day) had no adverse effect on rats' hematological and blood biochemical profiles. Nonetheless, decreased interleukin (IL)-6, and LDL-C levels, along with enhanced ferric ion reduction ability (FRA) and sulfhydryl content, were observed in the plasma of OSO-supplemented rats compared to the control and sunflower oil (SO) supplemented group. In addition, OSO supplementation stabilized apoA-I/HDL and augmented HDL-allied paraoxonase (PON)-1 activity. The microinjection of OSO-HDL (10 nL, 2 mg/mL) efficiently prevented the CML (500 ng)-induced zebrafish embryo mortality and developmental deformities. Similarly, OSO-HDL thwarted CML-posed neurotoxicity and demonstrated a significant hepatoprotective effect against CML-induced fatty liver changes, hepatic inflammation, oxidative stress, and apoptosis, as well as exhibiting a noticeable influence to revert CML-induced dyslipidemia. Conclusively, OSO supplementation demonstrated no toxic effects on rats, ameliorated plasma antioxidant status, and positively influenced HDL stability and functionality, leading to a protective effect against CML-induced toxicity in zebrafish.

Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities.

CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL2 and HDL3 remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu2+-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.

Cuban Policosanol (Raydel®) Exerts Higher Antioxidant and Anti-Glycation Activities than Chinese Policosanol (BOC Sciences) in Reconstituted High-Density Lipoproteins: In Vivo Anti-Inflammatory Activities in Zebrafish and Its Embryos.

The present study compares sugarcane-wax purified policosanols sourced from Cuba (Raydel®) and China (BOC Sciences) and utilized following the synthesis of reconstituted high-density lipoproteins (rHDL). The two policosanols exhibited distinctly different ingredient ratios of long-chain aliphatic alcohols, particularly 1-octacosanol (C28) and 1-tetratriacotanol (C34). After synthesizing rHDL with apolipoprotein A-I (apoA-I), the two policosanols bound well with phospholipid and apoA-I to form the discoidal rHDL. Notably, rHDL-1, containing Cuban policosanol, displayed the largest particle diameter at approximately 78 ± 3 nm. In contrast, both control rHDL (rHDL-0) and rHDL containing Chinese policosanol (rHDL-2) exhibited smaller particles, with diameters of approximately 58 ± 3 nm and 61 ± 2 nm, respectively. Furthermore, rHDL-1 demonstrated enhanced anti-glycation activity, safeguarding apoA-I from degradation within HDL, and displayed the antioxidant ability to inhibit LDL oxidation. A microinjection of each rHDL into zebrafish embryos in the presence of carboxymethyllysine (CML) revealed rHDL-1 to have the strongest antioxidant activity with the highest embryo survivability and normal developmental morphology. Dermal application to recover the wound revealed rHDL-1 to have the highest wound-healing activity (75%) and survivability (92%) in the cutaneous wound area in the presence of CML. In adult zebrafish, injecting CML (250 µg) caused acute death and hyperinflammation, marked by heightened neutrophil infiltration and interleukin (IL)-6 production in liver. However, co-administering rHDL-1 notably increased survival (85%) and exhibited strong anti-inflammatory properties, reducing IL-6 production while improving the blood lipid profile. However, a co-injection of rHDL-2 resulted in the lowest survivability (47%) with more hepatic inflammation. In conclusion, Cuban policosanol (Raydel®) has more desirable properties for the in vitro synthesis of rHDL with stronger anti-glycation and antioxidant activities than those of Chinese policosanol (BOC Sciences). Moreover, Raydel-policosanol-integrated rHDL demonstrates a noteworthy effect on accelerated wound healing and robust anti-inflammatory properties, leading to increased survivability in zebrafish embryos and adults by effectively suppressing CML-induced hyperinflammation.

Long-Term Supplementation of Royal Jelly (Raydel®) Improves Zebrafish Growth, Embryo Production and Survivability, Blood Lipid Profile and Functionality of Vital Organs: A 72-Weeks' Consumption Study.

Royal jelly is a honeybee product with substantial pharmacological and health promotional activities. Nevertheless, the health implications associated with the prolonged dietary supplementation of royal jelly have yet to be elucidated extensively. Herein, 72 weeks of dietary supplementation of royal jelly at 5% and 10% (w/w) were investigated to assess the impact on zebrafish survivability, body weight, liver, testis, ovary functionality, and blood lipid profile. The results revealed no adverse effect of 72 weeks of royal jelly supplementation on zebrafish survivability. Conversely, a noteworthy enhancement in the zebrafish body weight was observed in royal-jelly-supplemented zebrafish in a concentration-dependent manner [5% and 10% (w/w)]. Interestingly, female zebrafish were found to be more biased, with a significant 17% (p < 0.001) and 23% (p < 0.001) higher body weight enhancement after 72 weeks of consumption of 5% and 10% (w/w) royal jelly, compared to the male zebrafish. The histological outcome revealed no sign of hepatotoxicity; moreover, diminished reactive oxygen species (ROS) and apoptosis were observed in the hepatic tissue of the royal-jelly-supplemented group. Consistent with the histological outcomes, the liver function biomarkers, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), exhibited a significant decrease of 1.9-fold (p = 0.006) and 1.4-fold (p = 0.003) in zebrafish supplemented with royal jelly compared to those on a normal diet (ND) and zebrafish given supplements. Also, no sign of ovary and testis-related toxicity was observed in the royal-jelly-supplemented group during the 72-week period. Furthermore, the 10% (w/w) royal-jelly-consuming zebrafish exhibited a notable 2.1-fold increase (p = 0.018) in egg-laying ability compared to the ND-supplemented zebrafish. The 10% (w/w) royal jelly supplementation also effectively maintained the blood lipid profile by curtailing serum triglycerides (TG) and elevating high-density lipoprotein cholesterol (HDL-C). Conclusively, royal jelly dietary supplementation for a prolonged time found royal jelly to be safe to consume, to efficiently improve hepatic function, reproduction, and sexual health, and to augment the serum HDL-C level.

Synergistic Anti-Inflammatory Activity of Apolipoprotein A-I and CIGB-258 in Reconstituted High-Density Lipoproteins (rHDL) against Acute Toxicity of Carboxymethyllysine in Zebrafish and Its Embryo.

CIGB-258 is a 3 kDa altered peptide ligand from heat shock protein (HSP) 60 that exhibits anti-inflammatory activity against the acute toxicity of carboxymethyllysine (CML) with antioxidant and anti-glycation activities via protection of high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I). It is necessary to test a synergistic interaction between apoA-I and CIGB-258 in reconstituted high-density lipoproteins (rHDL). Several rHDLs were synthesized containing palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apoA-I, and CIGB-258 at molar ratios of 95:5:1:0, 95:5:1:0.1, 95:5:1:0.5, and 95:5:1:1 for rHDL-(1:0), rHDL-(1:0.1), rHDL-(1:0.5), and rHDL-(1:1), respectively. As the CIGB-258 content in rHDL was increased, the particle size of rHDL was 1.4-times higher than rHDL-(1:0) to rHDL-(1:1), from 60 nm to 83 nm, respectively. As the CIGB-258 content was increased, the rHDL showed the most resistance to isothermal denaturation by a urea treatment, and rHDL-(1:1) exhibited the highest structural stability and the strongest antioxidant ability against LDL oxidation. Co-treatment of rHDL-(1:0), rHDL-(1:0.5), and rHDL-(1:1) resulted in up to 10%, 24%, and 34% inhibition of HDL glycation, inhibition of HDL glycation, which was caused by the CML, with protection of apoA-I. Microinjection of each rHDL into zebrafish embryos in the presence of CML showed that a higher CIGB-258 content in rHDL was associated with higher survivability with the least inflammation and apoptosis. Furthermore, an intraperitoneal injection of rHDL and CML showed that a higher CIGB-258 content in rHDL was also associated with higher survivability of zebrafish and faster recovery of swimming ability. The rHDL-(1:1) group showed the lowest triglyceride, AST, and ALT serum levels with the least production of interleukin-6, oxidized product, and neutrophil infiltration in hepatic tissue. In conclusion, CIGB-258 could bind well to phospholipids and cholesterol to stabilize apoA-I in the rHDL structure against denaturation stress and larger particle sizes. The rHDL containing CIGB-258 enhanced the in vitro antioxidant ability against LDL oxidation, the anti-glycation activity to protect HDL, and the in vivo anti-inflammatory activity against CML toxicity in zebrafish adults and embryos. Overall, incorporating apoA-I and CIGB-258 in rHDL resulted in a synergistic interaction to enhance the structural and functional correlations in a dose-dependent manner of CIGB-258.

Ozonated Sunflower Oil (OSO) Alleviates Inflammatory Responses in Oxazolone-Induced Atopic Dermatitis (AD)-Like Mice and LPS-Treated RAW 264.7 Cells.

Ozone, a highly reactive oxidant molecule, is widely used as a complementary therapy for various skin diseases, including wound healing, pressure ulcers, diabetic foot, and infections. However, there is limited research on the effectiveness of ozone for atopic dermatitis (AD). Ozonated sunflower oil (OSO) is an active ingredient obtained from partially ozonated sunflower oil (SO). OSO markedly reduced the LPS-induced increase in IL-1ß and nitric oxide (NO) levels in RAW 264.7 mouse macrophage cells. Oxazolone (OXZ) was applied to hairless mice to induce AD-like skin symptoms and immune response. OSO significantly alleviated the OXZ-induced increases in the number of infiltrating mast cells, epidermal thickness, AD symptoms, thymic stromal lymphopoietin (TSLP), and filaggrin, as well as the serum levels of NO, IgE, IL-1ß, and TNF-α. Furthermore, OSO inhibited the IL-4/STAT3/MAPK pathway and the expression of NF-κB. Our results suggest that OSO treatment could relieve AD-mediated skin damage through its anti-inflammatory and antioxidant activities. Therefore, it can be used as a therapeutic agent against AD-related skin diseases.

Beeswax Alcohol Prevents Low-Density Lipoprotein Oxidation and Demonstrates Antioxidant Activities in Zebrafish Embryos and Human Subjects: A Clinical Study.

Oxidative stress is one of the primary instigators of the onset of various human ailments, including cancers, cardiovascular diseases, and dementia. Particularly, oxidative stress severely affects low-density lipid & protein (LDL) oxidation, leading to several detrimental health effects. Therefore, in this study, the effect of beeswax alcohol (BWA) was evaluated in the prevention of LDL oxidation, enhancement of paraoxonase 1 (PON-1) activity of high-density lipid & protein (HDL), and zebrafish embryo survivability. Furthermore, the implication of BWA consumption on the oxidative plasma variables was assessed by a preliminary clinical study on middle-aged and older human subjects (n = 50). Results support BWA augmentation of PON-1 activity in a dose-dependent manner (10-30 µM), which was significantly better than the effect exerted by coenzyme Q10 (CoQ10). Moreover, BWA significantly curtails LDL/apo-B oxidation evoked by CuSO4 (final 0.5 µM) and a causes a marked reduction in lipid peroxidation in LDL. The transmission electron microscopy (TEM) analysis revealed a healing effect of BWA towards the restoration of LDL morphology and size impaired by the exposure of Cu2+ ions (final 0.5 µM). Additionally, BWA counters the toxicity induced by carboxymethyllysine (CML, 500 ng) and rescues zebrafish embryos from development deformities and apoptotic cell death. A completely randomized, double-blinded, placebo-controlled preliminary clinical study on middle- and older-aged human subjects (n = 50) showed that 12 weeks of BWA (100 mg/day) supplementation efficiently diminished serum malondialdehyde (MDA) and total hydroperoxides and enhanced total antioxidant status by 25%, 27%, and 22%, respectively, compared to the placebo-control and baseline values. Furthermore, the consumption of BWA did not exhibit any noteworthy changes in physical variables, lipid profile, glucose levels, and biomarkers pertinent to kidney and liver function, thus confirming the safety of BWA for consumption. Conclusively, in vitro, BWA prevents LDL oxidation, enhances PON-1 activity in HDL, and positively influences oxidative variables in human subjects.

Prolonged Supplementation of Ozonated Sunflower Oil Bestows an Antiaging Effect, Improves Blood Lipid Profile and Spinal Deformities, and Protects Vital Organs of Zebrafish (Danio rerio) against Age-Related Degeneration: Two-Years Consumption Study.

Ozonated sunflower oil (OSO) is renowned for its diverse therapeutic benefits. Nonetheless, the consequences of extended dietary intake of OSO have yet to be thoroughly investigated. Herein, the effect of 2-year dietary supplementation of OSO was examined on the survivability, obesity, skeletal deformities, swimming behavior, and liver, kidney, ovary, and testis function of zebrafish. Results showed that the zebrafish feed supplemented with 20% (wt/wt) OSO for 2 years emerged with higher survivability and body weight management compared to sunflower oil (SO) and normal diet (ND)-supplemented zebrafish. Radio imaging (X-ray)-based analysis revealed 2.6° and 15.2° lower spinal curvature in the OSO-supplemented groups than in the SO and ND-supplemented groups; consistently, OSO-supplemented zebrafish showed better swimming behavior. The histology analysis of the liver revealed the least fatty liver change and interleukin (IL)-6 generation in the OSO-supplemented group. Additionally, a significantly lower level of reactive oxygen species (ROS), apoptotic, and senescent cells were observed in the liver of the OSO-supplemented zebrafish. Also, no adverse effect on the kidney, testis, and ovary morphology was detected during 2 years of OSO consumption. Moreover, lower senescence with diminished ROS and apoptosis was noticed in the kidney and ovary in response to OSO consumption. The OSO supplementation was found to be effective in countering age-associated dyslipidemia by alleviating total cholesterol (TC), triglycerides (TG), low-density lipoproteins (LDL-C) and elevating high-density lipoproteins (HDL-C)/TC levels. Conclusively, prolonged OSO consumption showed no adverse effect on the morphology and functionality of vital organs; in fact, OSO supplementation displayed a protective effect against age-associated detrimental effects on spinal deformities, vital organ functionality, cell senescence, and the survivability of zebrafish.

Combination Therapy of Cuban Policosanol (Raydel®, 20 mg) and Intensive Exercise for 12 Weeks Resulted in Improvements in Obesity, Hypertension, and Dyslipidemia without a Decrease in Serum Coenzyme Q10: Enhancement of Lipoproteins Quality and Antioxidant Functionality in Obese Participants.

Obesity and overweight, frequently caused by a lack of exercise, are associated with many metabolic diseases, such as hypertension, diabetes, and dyslipidemia. Aerobic exercise effectively increases the high-density lipoproteins-cholesterol (HDL-C) levels and alleviates the triglyceride (TG) levels. The consumption of Cuban policosanol (Raydel®) is also effective in enhancing the HDL-C quantity and HDL functionality to treat dyslipidemia and hypertension. On the other hand, no study has examined the effects of a combination of high-intensity exercise and policosanol consumption in obese subjects to improve metabolic disorders. In the current study, 17 obese subjects (average BMI 30.1 ± 1.1 kg/m2, eight male and nine female) were recruited to participate in a program combining exercise and policosanol (20 mg) consumption for 12 weeks. After completion, their BMI, waist circumference, total fat mass, systolic blood pressure (SBP), and diastolic blood pressure (DBP) reduced significantly up to around -15%, -13%, -33%, -11%, and -13%, respectively. In the serum lipid profile, at Week 12, a significant reduction was observed in the total cholesterol (TC) and triglyceride (TG) levels, up to -17% and -54% from the baseline, respectively. The serum HDL-C was elevated by approximately +12% from the baseline, as well as the percentage of HDL-C in TC, and HDL-C/TC (%), was enhanced by up to +32% at Week 12. The serum coenzyme Q10 (CoQ10) level was increased 1.2-fold from the baseline in all participants at Week 12. In particular, the male participants exhibited a 1.4-fold increase from the baseline. The larger rise in serum CoQ10 was correlated with the larger increase in the serum HDL-C (r = 0.621, p = 0.018). The hepatic function parameters were improved; the serum γ-glutamyl transferase decreased at Week 12 by up to -55% (p < 0.007), while the aspartate aminotransferase and alanine transaminase levels diminished within the normal range. In the lipoprotein level, the extent of oxidation and glycation were reduced significantly with the reduction in TG content. The antioxidant abilities of HDL, such as paraoxonase (PON) and ferric ion reduction ability (FRA), were enhanced significantly by up to 1.8-fold and 1.6-fold at Week 12. The particle size and number of HDL were elevated up to +10% during the 12 weeks, with a remarkable decline in the TG content, glycation extent, and oxidation. The improvements in HDL quality and functionality were linked to the higher survivability of adult zebrafish and their embryos, under the co-presence of carboxymethyllysine (CML), a pro-inflammatory molecule known to cause acute death. In conclusion, 12 weeks of Cuban policosanol (Raydel®, 20 mg) consumption with high-intensity exercise displayed a significant improvement in blood pressure, body fat mass, blood lipid profile without liver damage, CoQ10 metabolism, and renal impairment.

Enhancement of Antioxidant and Anti-Glycation Properties of Beeswax Alcohol in Reconstituted High-Density Lipoprotein: Safeguarding against Carboxymethyllysine Toxicity in Zebrafish.

The antioxidant and anti-inflammatory abilities of beeswax alcohol (BWA) are well reported in animal and human clinical studies, with a significant decrease in malondialdehyde (MDA) in the blood, reduced liver steatosis, and decreased insulin. However, there has been insufficient information to explain BWAs in vitro antioxidant and anti-inflammatory activity owing to its limited solubility in an aqueous buffer system. Herein, three distinct reconstituted high-density lipoproteins (rHDL) were prepared with palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apolipoprotein A-I (apoA-I), and BWA at molar ratios of 95:5:1:0 (rHDL-0), 95:5:1:0.5 (rHDL-0.5), and 95:5:1:1 (rHDL-1) and examined for antioxidant and anti-glycation effects. A rHDL containing BWA, precisely rHDL-1, displayed a remarkable anti-glycation effect against fructose (final 250 mM), induced glycation of HDL, and prevented proteolytic degradation of apoA-I. Also, BWA incorporated rHDL-0.5, and rHDL-1 displayed substantial antioxidant activity by inhibiting cupric ion-mediated low-density lipoprotein (LDL) oxidation. In contrast to rHDL-0, a 20 and 22% enhancement in ferric ion reduction ability (FRA) and paraoxonase (PON) activity was observed in HDL treated with rHDL-1, signifying the effect of BWA on the antioxidant activity enhancement of HDL. rHDL-1 efficiently inhibits Nε-carboxylmethyllysine (CML)-induced reactive oxygen species (ROS) generation and apoptosis in zebrafish embryos, consequently improving embryo survivability and developmental deformities impaired by the CML. The dermal application of rHDL-1 to the CML-impaired cutaneous wound of the adult zebrafish inhibited ROS production and displayed potent wound-healing activity. Conclusively, incorporating BWA in rHDL significantly enhanced the anti-glycation and antioxidant activities in rHDL via more stabilization of apoA-I with a larger particle size. The rHDL containing BWA facilitated the inherent antioxidant ability of HDL to suppress the CML-induced toxicities in zebrafish embryos and ameliorate CML-aggravated chronic wounds in adult zebrafish.

Cuban Policosanol (Raydel®) Potently Protects the Liver, Ovary, and Testis with an Improvement in Dyslipidemia in Hyperlipidemic Zebrafish: A Comparative Study with Three Chinese Policosanols.

Many policosanols from different sources, such as sugar cane and rice bran, have been marketed worldwide to improve blood lipid profiles. But so far, no comparative study has commenced elucidating the effect of different policosanols to improve the blood lipid profile and other beneficial effects. This study compared the efficacy of four different policosanols, including one sugar cane wax alcohol from Cuba (Raydel®) and three policosanols from China (Xi'an Natural sugar cane, Xi'an Realin sugar cane, and Shaanxi rice bran), to treat dyslipidemia in hyperlipidemic zebrafish. After 12 weeks of consumption of each policosanol (final 0.1% in diet, wt/wt) and a high-cholesterol diet (HCD, final 4%, wt/wt), the Raydel policosanol group and the Xi'an Natural policosanol group showed the highest survivability, of approximately 81%. In contrast, the Xi'an Realin policosanol and the Shaanxi policosanol groups showed 57% and 67% survivability, respectively. Among the five HCD groups, the Raydel policosanol group showed the lowest serum total cholesterol (TC, p < 0.001 versus HCD control) and triglyceride (p < 0.001 versus HCD control), with the highest percentage of high-density lipoproteins-cholesterol in TC. The Raydel policosanol group also showed the lowest serum aspartate aminotransferase and alanine aminotransferase levels, with the least infiltration of inflammatory cells and interleukin-6 production in hepatocytes with a marked reduction in reactive oxygen species (ROS) production and fatty liver changes. In the ovary, the Raydel policosanol group also showed the highest content of mature vitellogenic oocytes with the lowest production of reactive oxygen species and cellular apoptosis in ovarian cells. In the testes, the Raydel policosanol group also showed the healthiest morphology for spermatogenesis, with the lowest interstitial area and reactive oxygen species production in testicular cells. Conclusively, among the tested policosanols, Cuba (Raydel®) policosanol exhibited a comparatively better effect in maintaining zebrafish body weight, survivability, blood lipid profile, hepatic function biomarkers, fatty liver changes, ROS generation, inflammation, and restoration of the cell morphology in ovaries and testes affected by the HCD consumption.

Comparison of Policosanols via Incorporation into Reconstituted High-Density Lipoproteins: Cuban Policosanol (Raydel®) Exerts the Highest Antioxidant, Anti-Glycation, and Anti-Inflammatory Activity.

Reconstituted high-density lipoproteins (rHDL) containing each policosanol from Cuba (Raydel®), China (Shaanxi Pioneer), and the United States (Lesstanol®) were synthesized to compare the physiological properties of policosanol depending on sources and origin countries. After synthesis with apolipoproteinA-I (apoA-I) into rHDL, all policosanols bound well with phospholipid and apoA-I to form discoidal rHDL. An rHDL containing Cuban policosanol (rHDL-1) showed the largest rHDL particle size of around 83 ± 3 nm, while rHDL containing Chinese policosanol (rHDL-2) or American policosanol (rHDL-3) showed smaller particles around 63 ± 3 nm and 60 ± 2 nm in diameter, respectively. The rHDL-1 showed the strongest anti-glycation activity to protect the apoA-I degradation of HDL from fructose-mediated glycation: approximately 2.7-times higher ability to suppress glycation and 1.4-times higher protection ability of apoA-I than that of rHDL-2 and rHDL-3. The rHDL-1 showed the highest antioxidant ability to inhibit cupric ion-mediated LDL oxidation in electromobility and the quantification of oxidized species. A microinjection of each rHDL into a zebrafish embryo in the presence of carboxymethyllysine (CML) showed that rHDL-1 displayed the strongest anti-oxidant activity with the highest embryo survivability, whereas rHDL-2 and rHDL-3 showed much weaker protection ability, similar to rHDL alone (rHDL-0). An intraperitoneal injection of CML (250 µg) into adult zebrafish caused acute death and hyperinflammation with an elevation of infiltration of neutrophils and IL-6 production in the liver. On the other hand, a co-injection of rHDL-1 resulted in the highest survivability and the strongest anti-inflammatory ability to suppress IL-6 production with an improvement of the blood lipid profile, such as elevation of HDL-C and lowering of the total cholesterol, LDL-cholesterol, and triglyceride. In conclusion, Cuban policosanol exhibited the most desirable properties for the in vitro synthesis of rHDL with the stabilization of apoA-I, the largest particle size, anti-glycation against fructation, and antioxidant activities to prevent LDL oxidation. Cuban policosanol in rHDL also exhibited the strongest in vivo antioxidant and anti-inflammatory activities with the highest survivability in zebrafish embryos and adults via the prevention of hyperinflammation in the presence of CML.

Ozonated Sunflower Oil Exerted Potent Anti-Inflammatory Activities with Enhanced Wound Healing and Tissue Regeneration Abilities against Acute Toxicity of Carboxymethyllysine in Zebrafish with Improved Blood Lipid Profile.

Ozonated sunflower oil (OSO) is an established therapeutic agent and nutraceutical harboring various therapeutic values, including antiallergic, derma-protective, and broad-spectrum antimicrobial activity. Conversely, the medicinal aspects of OSO for wound healing, tissue regeneration, and treatment of inflammation in dyslipidemia have yet to be fully elucidated. Herein, a comparative effect of OSO and sunflower oil (SO) was investigated to heal cutaneous wound and tissue regeneration of zebrafish impediment by carboxymethyllysine (CML) toxicity, following impact on hepatic inflammation and blood lipid profile. After OSO (final 2%, 1 µL) and SO's (final 2%, 1 µL) treatment, substantial healing was elicited by OSO in the cutaneous wound of zebrafish impaired by CML (final 25 µg). As an important event of wound healing, OSO scavenges the reactive oxygen species (ROS), rescues the wound from oxidative injury, and triggers the essential molecular events for the wound closer. Furthermore, the intraperitoneal injection of OSO was noted to counter the CML-induced adversity and prompt tissue regeneration in the amputated tail fin of zebrafish. Additionally, OSO counters the CML-induced neurotoxicity and rescues the zebrafish from acute mortality and paralysis, along with meticulous recovery of hepatic inflammation, fatty liver changes, and diminished ROS and proinflammatory interleukin (IL)-6 production. Moreover, OSO efficiently ameliorated CML-induced dyslipidemia by alleviating the total blood cholesterol (TC), triglyceride (TG), and increasing high-density lipoproteins cholesterol (HDL-C). The outcome of multivariate assessment employing principal component analysis and hierarchical cluster analysis supports a superior therapeutic potential of OSO over SO against the clinical manifestation of CML. Conclusively, OSO owing to its antioxidant and anti-inflammatory potential, counters CML-induced toxicity and promotes wound healing, tissue regeneration, hepatoprotection, improved blood lipid profile, and survivability of zebrafish.

Long-Term Supplementation of Ozonated Sunflower Oil Improves Dyslipidemia and Hepatic Inflammation in Hyperlipidemic Zebrafish: Suppression of Oxidative Stress and Inflammation against Carboxymethyllysine Toxicity.

Ozonated sunflower oil (OSO) is a well-known functional oil with antioxidant, antimicrobial, anti-allergic, and skin-moisturizing properties. However, studies on the effects of OSO on high-cholesterol diet (HCD)-induced metabolic disorders have been scarce. In the current study, we aimed to determine the anti-inflammatory effects of OSO on lipid metabolism in adult hypercholesterolemic zebrafish and its embryos. Microinjection of OSO (final 2%, 10 nL) into zebrafish embryos under the presence of carboxymethyllysine (CML, 500 ng) protected acute embryo death up to 61% survival, while sunflower oil (final 2%) showed much less protection at around 42% survival. The microinjection of OSO was more effective than SO to inhibit reactive oxygen species (ROS) production and apoptosis in the CML induced embryo toxicity. Intraperitoneal injection of OSO under the presence of CML protected acute death from CML-induced neurotoxicity with improved hepatic inflammation, less detection of ROS and interleukin (IL)-6, and lowering blood total cholesterol (TC) and triglyceride (TG), while the SO-injected group did not protect the CML-toxicity. Long-term supplementation of OSO (final 20%, wt/wt) with HCD for 6 months resulted in higher survivability than the HCD alone group or HCD + SO group (final 20%, wt/wt) with significant lowering of plasma TC and TG levels. The HCD + OSO group showed the least hepatic inflammation, fatty liver change, ROS, and IL-6 production. In conclusion, short-term treatment of OSO by injection exhibited potent anti-inflammatory activity against acute neurotoxicity of CML in zebrafish and their embryo. Long-term supplementation of OSO in the diet also revealed the highest survivability and blood lipid-lowering effect through potent antioxidant and anti-inflammatory activity.

Protection of Liver Functions and Improvement of Kidney Functions by Twelve Weeks Consumption of Cuban Policosanol (Raydel®) with a Decrease of Glycated Hemoglobin and Blood Pressure from a Randomized, Placebo-Controlled, and Double-Blinded Study with Healthy and Middle-Aged Japanese Participants.

Policosanol consumption has been associated with treating blood pressure and dyslipidemia by increasing the level of high-density lipoproteins-cholesterol (HDL-C) and HDL functionality. Although policosanol supplementation also ameliorated liver function in animal models, it has not been reported in a human clinical study, particularly with a 20 mg doage of policosanol. In the current study, twelve-week consumption of Cuban policosanol (Raydel®) significantly enhanced the hepatic functions, showing remarkable decreases in hepatic enzymes, blood urea nitrogen, and glycated hemoglobin. From the human trial with Japanese participants, the policosanol group (n = 26, male 13/female 13) showed a remarkable decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from baseline up to 21% (p = 0.041) and 8.7% (p = 0.017), respectively. In contrast, the placebo group (n = 26, male 13/female 13) showed almost no change or slight elevation. The policosanol group showed a 16% decrease in γ-glutamyl transferase (γ-GTP) at week 12 from the baseline (p = 0.015), while the placebo group showed a 1.2% increase. The policosanol group exhibited significantly lower serum alkaline phosphatase (ALP) levels at week 8 (p = 0.012), week 12 (p = 0.012), and after 4-weeks (p = 0.006) compared to those of the placebo group. After 12 weeks of policosanol consumption, the ferric ion reduction ability and paraoxonase of serum were elevated by 37% (p < 0.001) and 29% (p = 0.004) higher than week 0, while placebo consumption showed no notable changes. Interestingly, glycated hemoglobin (HbA1c) in serum was lowered significantly in the policosanol group 4 weeks after consumption, which was approximately 2.1% (p = 0.004) lower than the placebo group. In addition, blood urea nitrogen (BUN) and uric acid levels were significantly lower in the policosanol group after 4 weeks: 14% lower (p = 0.002) and 4% lower (p = 0.048) than those of the placebo group, respectively. Repeated measures of ANOVA showed that the policosanol group had remarkable decreases in AST (p = 0.041), ALT (p = 0.008), γ-GTP (p = 0.016), ALP (p = 0.003), HbA1c (p = 0.010), BUN (p = 0.030), and SBP (p = 0.011) from the changes in the placebo group in point of time and group interaction. In conclusion, 12 weeks of 20 mg consumption of policosanol significantly enhanced hepatic protection by lowering the serum AST, ALT, ALP, and γ-GTP via a decrease in glycated hemoglobin, uric acid, and BUN with an elevation of serum antioxidant abilities. These results suggest that improvements in blood pressure by consumption of 20 mg of policosanol (Raydel®) were accompanied by protection of liver function and enhanced kidney function.

Aging and chronic inflammation: highlights from a multidisciplinary workshop.

Aging is a gradual, continuous series of natural changes in biological, physiological, immunological, environmental, psychological, behavioral, and social processes. Aging entails changes in the immune system characterized by a decrease in thymic output of naïve lymphocytes, an accumulated chronic antigenic stress notably caused by chronic infections such as cytomegalovirus (CMV), and immune cell senescence with acquisition of an inflammatory senescence-associated secretory phenotype (SASP). For this reason, and due to the SASP originating from other tissues, aging is commonly accompanied by low-grade chronic inflammation, termed "inflammaging". After decades of accumulating evidence regarding age-related processes and chronic inflammation, the domain now appears mature enough to allow an integrative reinterpretation of old data. Here, we provide an overview of the topics discussed in a recent workshop "Aging and Chronic Inflammation" to which many of the major players in the field contributed. We highlight advances in systematic measurement and interpretation of biological markers of aging, as well as their implications for human health and longevity and the interventions that can be envisaged to maintain or improve immune function in older people.